Our objective is to define cellular and molecular mechanisms by which airway smooth muscle cells contribute to airway hyperreactivity. Initial studies focused on acute, postjunctional events that increase smooth muscle hyperresponsiveness. New studies are proposed to define the roles of ERK and p38/RK mitogen-activated protein kinases (MAP kinases) in canine tracheal smooth muscle contraction and airway wall remodeling. The hypothesis is that MAP kinases are components of protein kinase cascades controlling contractile protein function, contractile protein gene expression and structure of the cytoskeleton. Coupling of ERK MAP kinases to contractile system function will be studied by: i) Assaying agonist and Ca2+-dependence of ERK MAP kinase activation, ii) Determining the functional consequences of ERK activation, and iii) Sequencing ERK isoforms expressed in differentiated canine tracheal smooth muscle. Coupling of ERK MAP kinases to contractile protein gene expression in cultured tracheal myocytes will be investigated by: i) Determining whether chronic receptor activation regulates both ERK MAP kinase and contractile protein expression, and iii) Test the necessity of MAP kinases in maintaining the contractile phenotype by inhibiting MAP kinase expression with antisense oligodeoxynucleotides. Coupling of p38/RK MAP kinase to cytoskeletal structure may occur through activation of MAP kinase-activated kinase-II (MAPKAP II) which phosphorylates the 27 kDa heat shock protein (HSP27). To test this hypothesis we will: i) Assay tyrosine phosphorylation of p38/RK MAP kinase, activation of MAPKAP II and phosphorylation of HSP27, ii) Establish actin binding activity of HSP27, iii) Determine whether treatments that increase HSP27 phosphorylation also increase actin polymerization, and iv) Investigate the function of HSP27 in migration and shortening of cultured myocytes. We propose to define the roles of ERK and p38/RK MAP kinases in contractile and proliferative phenotypes of airway smooth muscle, which may be valuable in designing strategies to inhibit or reverse smooth muscle hyperreactivity and airway wall remodeling that occurs in asthma.